Phosphodiesterase-4 (PDE4), an enzyme catalyzing the breakdown of cyclic AMP (cAMP), has been implicated in memory impairment associated with Alzheimer's disease (AD). Inhibition of PDE4 increases intracellular cAMP and subsequently activates its downstream target cAMP-responsive-element-binding protein (CREB). Primarily through this mechanism, the PDE4 inhibitor rolipram not only counteracts the inhibitory effects of high levels of amyloid-beta peptide (Abeta) on cAMP signaling and memory, but also delays the natural progression of Abeta-induced synaptic and cognitive abnormalities. These point to PDE4 as a potential target for the treatment of memory deficits in AD. Based on recent data from our laboratory and others, we hypothesize that, among the four PDE4 subtypes (PDE4-A, B, C, and D), PDE4D plays a major role in the mediation of memory loss associated with AD. Thus, inhibition or knockdown of PDE4D should result in reversal of Abeta-induced cAMP/CREB inhibition and memory deficits. The primary objective of this proposal is to test this hypothesis in a rodent model. For this purpose, the following specific aims are proposed. First, determine whether knockdown of PDE4D reverses Abeta-induced inhibition of cAMP/CREB signaling in vitro. This will be accomplished by detecting levels of cAMP and phospho-CREB (pCREB) in primary cultures of hippocampal neurons treated with Abeta1-42 (Abeta42) and lentiviral vectors expressing microRNAs (miRNAs) of PDE4D4 and PDE4D5, the primary PDE4D variants in the hippocampus. Second, determine whether PDE4D knockdown in the hippocampus reverses Abeta- induced deficits of cAMP signaling and memory. This will be accomplished by examining pCREB levels in the hippocampus in vivo and memory performance using the Morris water-maze task in rats, which are administered Abeta42 following the knockdown of PDE4D by miRNAs. Gene silencing will be carried out by microinfusions of lenti-PDE4D4/5-miRNAs into bilateral CA1 subregions of the rat hippocampus. Successful completion of these experiments will lead to a better understanding of the role of PDE4D in cAMP signaling and the determination of the contribution of PDE4D to memory deficits associated with AD. Using miRNA gene silencing, a powerful and unique technique for identifying gene functions, will make this happen and aid and encourage the development of PDE4 subtype-selective inhibitors as novel treatments for AD, which affects nearly 5 million Americans. This is an issue of high relevance to public health. Since knockdown of a specific PDE4 subtype has never been investigated in neurons, this project involves considerable risk such as the possible ineffectiveness of PDE4D-miRNAs. Nevertheless, it may lead to an extensive exploration of functions of PDE4 subtypes and even their 22 splice variants by the innovative use of gene silencing. The outcomes of this project could lead to a breakthrough in the PDE research area and have major impact on research of PDE4, a potential target for treatment of neurodegenerative disorders such as AD. PUBLIC HEALTH RELEVANCE: It has been found that an enzyme called phosphodiesterase-4 (PDE4), which helps in the breakdown of the important intracellular second messenger cyclic AMP (cAMP), plays an important role in mediating memory regulation in Alzheimer's disease (AD), a neurodegenerative disorder characterized by progressive loss of memory and commonly found in people over age 65. We propose experiments to find out whether reducing activity of one form, PDE4D, in the rat hippocampus reverses memory deficits and cAMP signaling decreases induced by misfolded proteins called beta amyloid (Abeta), which critically contributes to memory loss in AD. If these experiments are successful, they will not only help understand the important contribution of PDE4D to cAMP signaling and memory associated with AD, but encourage and accelerate the development of selective blockers of PDE4D for treating memory loss in AD, which affects approximately 20 million people worldwide and nearly 5 million people in the United States.